Isoprenoid-chained lipid β-XylOC16+4—A novel molecule for in meso membrane protein crystallization

doi:10.1016/j.jcrysgro.2010.08.018

Journal of Crystal Growth

Volume 312, Issue 22, 1 November 2010, Pages 3326-3330

https://doi.org/10.1016/j.jcrysgro.2010.08.018 Get rights and content

Abstract

Here we report a successful use of a recently developed isoprenoid-chained lipid family for in meso crystallization of membrane proteins. The isoprenoid-chained lipid 1-O-(3,7,11,15-tetramethylhexadecyl)-β–d-xyloside (β-XylOC₁₆₊₄) used as a host lipid for in meso crystallization provided high quality bacteriorhodopsin (bR) crystals (P6₃ space group) diffracting to high resolution and characterized by low twinning ratio. β-XylOC₁₆₊₄ has an isoprenoid chain with methyl branches at each 4th position and a xylose group in the water-soluble part. These peculiarities make the lipid clearly distinguishable in the bR crystalline lattice and provides a unique opportunity to study the role of the host lipid in the in meso crystallization. We conclude that β-XylOC₁₆₊₄ may have a general application for in meso crystallization for a wide range of membrane proteins. The cubic phase of β-XylOC₁₆₊₄ is present over a wide range of temperatures and is stable at low temperature (down to about 8 °C). This opens up the possibility of using temperature as a tool for the optimization of in meso crystallization with additional advantages for the crystallization of membrane proteins at lower temperatures where the proteins of interest may be more stable.

Introduction

Membrane proteins are the main functional units of membranes and represent roughly one-third of the proteins encoded in the genome. In spite of the fact that approximately 70% of drugs have membrane proteins as their target, only about 1% of structures in the Protein Data Bank are membrane proteins. X-ray protein crystallography is one of the most powerful tools to determine protein structure and to provide the basis for understanding molecular mechanisms of protein function. However, high resolution X-ray diffraction studies require well ordered protein crystals, but unfortunately crystallization of membrane proteins is still a challenge. One of the most promising methods to overcome this challenge is the in meso crystallization approach, where the lipid cubic or sponge phase is used as a crystallization matrix. Recently, this method was demonstrated to be applicable to different membrane proteins including G protein-coupled receptors [1], membrane protein complexes [2], [3] and bacteriorhodopsin (bR) [4], which for a long time failed to be crystallized by the in surfo method. For the in meso crystallization 1-oleoyl-rac-glycerol (monoolein, MO) is generally used to create a bicontinuous lipid bilayer crystallization matrix.

Here we present our results of the successful use of a new lipid – 1-O-(3,7,11,15-tetramethylhexadecyl)-β-d-xyloside (β-XylOC₁₆₊₄) – for bR in meso crystallization. Usually bR crystals obtained by in meso crystallization are perfectly twinned, which significantly complicates structural analysis and is probably one of the main reasons for controversial mechanisms of bR function [5]. Hexagonal plate bR crystals grown in the new lipid mesophase diffracted well and had zero or low twinning ratio. Due to the characteristic molecular shape of β-XylOC₁₆₊₄ it was possible to identify the volume occupied by the host lipid in the crystal structure.

Section snippets

Synthesis and purification of β-XylOC₁₆₊₄

β-XylOC₁₆₊₄ was synthesized as described in the previous papers [6], [7], [8], [9], [10] and purified as in [11]. The β-anomeric purity of the sample checked by NMR, gas chromatography and TLC was at least 99.3%.

bR expression, purification and crystallization

Purple membranes were extracted from Halobacterium salinarium S9 [12]. bR was solubilized in n-octyl-β-d-glucoside (OG) and purified as described in detail in [13] and crystallized in the lipidic meso phase formed by β-XylOC₁₆₊₄. The protein was mixed with lipid in the ratio 1 μl of 10

bR crystallization in β-XylOC₁₆₊₄

There are a lot of compounds that can be screened to improve the number of successful membrane protein in meso crystallization trials such as lipids, detergents, small molecule additives and traditional crystallization reagents, etc. Using different classes of known detergents (with different combinations of both polar headgroups and hydrophobic chain lengths) it is possible to choose the detergent most suitable for each particular membrane protein target [21]. Unfortunately this is not the

Conclusions

The isoprenoid-chained lipid β-XylOC₁₆₊₄ used as a host lipid for in meso crystallization provided high quality bR crystals diffracting to high resolution and further characterized by low twinning ratio. β-XylOC₁₆₊₄ has a characteristic shape, which makes it clearly distinguishable in the membrane protein crystalline lattice, where it is shown to stabilize protein contacts. We suppose that β-XylOC₁₆₊₄ may have a general application for in meso crystallization of a wide range of membrane

Acknowledgements

We are thankful to K.-E. Jaeger for the support of this work and C. Baeken for the help with bR production.

We would like to thank the ESRF and ESRF Structural Biology Group for the opportunity and help with data collection.

This work was financially supported in part by an NEDO International Joint Research Program (Subject: Molecular device for hydrogen production), the AIST Basic Research Program (Subject: Studies on bionanomaterials), the Targeted Proteins Research Program (TPRP) from Ministry

References (31)

S. Hanessian et al.
Chemistry of the glycosidic linkage.O-glycosylations catalyzed by stannic chloride, in the d-ribofuranose and d-glucopyranose series
Carbohydr. Res.
(1977)
H. Minamikawa et al.
Synthesis of 1,3-di-O-alkyl-2-O-(beta-glycosyl)glycerols bearing oligosaccharides as hydrophilic groups
Chem. Phys. Lipids
(1994)
D. Oesterhelt et al.
Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane
Methods Enzymol.
(1974)
B. Schobert et al.
Crystallographic structure of the K intermediate of bacteriorhodopsin: conservation of free energy after photoisomerization of the retinal
J. Mol. Biol.
(2002)
H. Luecke et al.
Structure of bacteriorhodopsin at 1.55 angstrom resolution
J. Mol. Biol.
(1999)
A.M. Seddon et al.
Membrane proteins, lipids and detergents: not just a soap opera
Biochim. Biophys. Acta
(2004)
Y. Misquitta et al.
Rational design of lipid for membrane protein crystallization
J. Struct. Biol.
(2004)
L.V. Misquitta et al.
Membrane protein crystallization in lipidic mesophases with tailored bilayers
Structure
(2004)
W. Shinoda et al.
Dynamics of a highly branched lipid bilayer: a molecular dynamics study
Chem. Phys. Lett.
(2004)
H. Qiu et al.
The phase diagram of the monoolein/water system: metastability and equilibrium aspects
Biomaterials
(2000)

H. Belrhali et al.

Protein, lipid and water organization in bacteriorhodopsin crystals: a molecular view of the purple membrane at 1.9 A resolution

Structure

(1999)

V. Cherezov et al.

High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor

Science

(2007)

V.I. Gordeliy et al.

Molecular basis of transmembrane signalling by sensory rhodopsin II-transducer complex

Nature

(2002)

R. Moukhametzianov et al.

Development of the signal in sensory rhodopsin and its transfer to the cognate transducer

Nature

(2006)

E. Pebay-Peyroula et al.

X-ray structure of bacteriorhodopsin at 2.5 angstroms from microcrystals grown in lipidic cubic phases

Science

(1997)

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Isoprenoid-chained lipid β-XylOC16+4—A novel molecule for in meso membrane protein crystallization

Abstract

Introduction

Section snippets

Synthesis and purification of β-XylOC16+4

bR expression, purification and crystallization

bR crystallization in β-XylOC16+4

Conclusions

Acknowledgements

Carbohydr. Res.

Chem. Phys. Lipids

Methods Enzymol.

J. Mol. Biol.

J. Mol. Biol.

Biochim. Biophys. Acta

J. Struct. Biol.

Structure

Chem. Phys. Lett.

Biomaterials

Structure

High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor

Science

Molecular basis of transmembrane signalling by sensory rhodopsin II-transducer complex

Nature

Development of the signal in sensory rhodopsin and its transfer to the cognate transducer

Nature

X-ray structure of bacteriorhodopsin at 2.5 angstroms from microcrystals grown in lipidic cubic phases

Science

Isoprenoid-chained lipid β-XylOC₁₆₊₄—A novel molecule for in meso membrane protein crystallization

Synthesis and purification of β-XylOC₁₆₊₄

bR crystallization in β-XylOC₁₆₊₄